Companion animal feed with combination of 25-hydroxyvitamin d and antioxidants/anti-inflammatories

ABSTRACT

This invention relates to the use of the combination of 25-hydroxyvitamin D3 (“25-OH D3”) and antioxidants/anti-inflammatories (ascorbic acid vitamin E and canthaxanthin) to make a premix or feed which can ameliorate various problems observed in companion animals which have been subject to overfeeding. Foods containing the 25-OH D3 and antioxidants/anti-inflammatories and premixes are also provided.

BRIEF DESCRIPTION OF THE INVENTION

This invention relates to the combination of 25-hydroxyvitamin D (“25-OHD3” and/or “25-OH D2”) and antioxidants/anti-inflammatories (ascorbicacid, Vitamin E and a carotenoid) for use in food for companion animals,especially cats and dogs. This combination of nutritional supplementsprotects against various adverse effects associated with hyperphagia andrelated obesity. It also relates to feed and feed premixes for companionanimals containing the combination of 25-hydroxyvitamin D and theantioxidants/anti-inflammatories.

BACKGROUND OF THE INVENTION

Hyperphagia and therefore adiposity can occur in companion animalsincluding dogs and cats. Overfeeding-induced diseases which can resultinclude metabolic disorders including diabetes and cardiovascularproblems. Pet owners do not always regulate the volume of food which isoffered to the animal, and some animals will eat past the point ofsatiation.

In poultry which is used as a model, elevated concentrations of plasmaglucose, non-esterified fatty acids, very low density lipoprotein,triacylglycerol, phospholipids, ceramide and sphingomyelin were observedduring hyperphagia. Furthermore, hepatic and circulating ceramide andsphingomyelin accumulation, and up-regulation of pro-inflammatory IL-1βexpression in liver and adipose tissues systemically manifested thedevelopment of lipotoxicity in feed-satiated hens. Ceramide is a keyintermediate linking certain nutrients (i.e. saturated fats) andinflammatory cytokines (e.g. tumor necrosis factor-α, TNFα) to theregulation of cell function and antagonizing insulin signaling andmitochondrial function. Moreover, as a result of its toxic effects onparticularly susceptible cell types, ceramide has the capacity to damagethe heart, pancreas, and vasculature. Lipotoxicity leading to impairedovarian dysfunctions, including follicle atresia, ovarian regression,and a decline of circulating estradiol levels in feed-satiated hens, wasfurther exemplified by ceramide accumulation and up-regulation of IL-1β,serine palmitoyltransferase, and sphingomyelinase transcript abundance,but suppressed protein kinase Akt activation within the hierarchicalfollicles. In vivo evidence has thus delineated the actions of ceramideand IL-1β in mediating overfeeding-induced follicle atresia andprogression of ovarian involution in broiler hens.

Hy⋅D® (registered trademark for 25-OH-D3; available from DSM NutritionalProducts, Switzerland has been used to promote bone and joint health incompanion animals, particularly large breed dogs.

The combination of 25-OH D3 and canthaxanthin has also been used inpoultry. WO2010/057811 (DSM IP ASSETS, BV) describes this combinationfor use in improving hatchability, fertility, and lower embryo mortalityin poultry. The combination is commercially available under thetrademark MAXICHICK. There is no mention in the patent publication ofthe inclusion of ascorbic acid and high vitamin E levels, nor the usesto ameliorate the adverse effects of hyperphagia-related obesity.

WO14/191153 (DSM IP ASSETS B.V) teaches the combination of canthaxanthinand at least one of Vitamin C, Vitamin E, selenium, and optionally atleast one of thymol, eugenol, vanillin and gamma-terpinene can improveimmune statues, bone health, skeletal development and growth and feedconversion, particularly when flocks are subject to stress associatedwith vaccination.

Vitamin C (ascorbic acid) is often not included as a supplement in someanimal diets, as the many animals can produce sufficient Vitamin C undernormal rearing conditions. However, it has been used in some specificconditions, such as in heat stress situations.

Vitamin E is generally added to companion animal food. For dogs therecommended minimum amounts range from 36-50 IU/kg on a dry matterbasis. For cats, the minimum is from 38-50.7 IU/kg, also on a dry matterbasis. Many premium pet foods have a much higher amount of Vitamin E,and it can be as high as 500 IU/kg.

There is a need to reduce or ameliorate the metabolic and hormonaldysregulations as a result of hyperphagia in companion animals in orderto ameliorate, prevent or treat impaired glucose regulation, insulinactions, cardiomyopathies, and ultimately ascites and sudden death.

DETAILED DESCRIPTION OF THE INVENTION

It has been found, accordance with this invention that the combinationof 25-hydroxyvitamin D (25-OH D3 and/or 25-OH D2) andantioxidants/anti-inflammatories ameliorates adverse metabolicconditions observed when hens are fed ad libitum. The hen was uses as amodel for ad libitum fed companion animals such as a cat or dog at theyalso can experience hyperphagia-related obesity. It has also been foundin accordance with this invention, that the bio-actives traditionallyincluded in the food as antioxidants also possess an anti-inflammatoryactivity which mitigates the adverse effects of hyperphagia-relatedobesity.

It has also been found that the combination of 25-OH D andantioxidants/anti-inflammatories can ameliorate, reduce the severity of,or prevent cardiovascular problems related to hyperphagia.

As 25-OH D2 and 25-OH D3 may act in a similar fashion afteradministration, it is envisioned that either may be used separately incombination with antioxidants/anti-inflammatories, or a mixture of both25-OH D3 and 25-OH D2 may be used in combination withantioxidants/anti-inflammatories. If used together, the ratio of 25-OHD3: 25-OH D2 is not a critical part of the invention. 25-OH D3 usedalone is preferred.

The antioxidants/anti-inflammatories of this invention comprise thecombination of ascorbic acid, vitamin E and a carotenoid. Thus oneaspect of this invention is the combination of 25-OH D3, a carotenoid,vitamin E and ascorbic acid. Another embodiment is the combination of25-OHD2 a carotenoid, Vitamin E and ascorbic acid. Another embodiment isthe combination of 25-OHD3, 25-OHD2, a carotenoid, Vitamin E andascorbic acid. The carotenoid is at least one selected from the groupconsisting of: lycopene, astaxanthin, cryptoxanthin, beta-carotene,lutein, zeaxanthin and canthaxanthin. Most preferred is beta carotene.

Another aspect of this invention is the combination of

-   -   a) 25-OH D,    -   b) a carotenoid selected from the group consisting of: lycopene,        astaxanthin, cryptoxanthin, beta-carotene, lutein, zeaxanthin        and canthaxanthin,    -   c) vitamin E    -   d) ascorbic acid, and    -   e) optionally further comprises at least one further bio-active        ingredient selected from the group consisting of:

Vitamin D, Vitamin B2, Vitamin B6, Niacin, Zinc, Copper, Manganese, andSelenium. Preferably the 25-OH D is 25-OH D3. Preferably at leastVitamin D is a further bio-active ingredient. Sometimes the furtherbio-active ingredients include at least Vitamin D and Selenium. In somecases, all the further bio-active ingredients are added.

Another aspect of this invention is a companion animal food comprisingthe combination of 25-OH D2 or 25-OH D3 or mixtures thereof, ascorbicacid, Vitamin E and a carotenoid, preferably beta carotene.

Yet another embodiment is a companion animal food comprising thecombination of 25-OH D, a carotenoid, preferably beta carotene, vitaminE and ascorbic acid, which optionally further comprises at least onefurther bio-active ingredient selected from the group consisting of:Vitamin D, Vitamin B2, Vitamin B6, Niacin, Zinc, Copper, Manganese,Selenium and combinations thereof. Preferably the 25-OH D is 25-OH D3.Sometimes the further bio-active ingredients include at least Vitamin Dand Selenium. In some cases, all the further bio-active ingredients areadded.

Another aspect of this invention are premixes for a companion animalfood which comprise the combination of 25-OH D, vitamin E, ascorbic acidand a carotenoid, preferably beta carotene. Preferably, the 25-OH D is25-OH D3. The premixes and subsequent feed, when fed to an animal proneto hyperphagia or obesity improves/ameliorates adverse metabolicconditions associated with hyperphagia. In some embodiments the feed andpremix also comprises at least one further bio-active ingredientselected from the group consisting of:

-   -   Vitamin D, Vitamin B2, Vitamin B6, Niacin, Zinc, Copper,        Manganese, Selenium and combinations thereof. Sometimes the        further bio-active ingredients include at least Vitamin D and        Selenium. In some cases, all the further bio-active ingredients        are added.

Another aspect of this invention is a method of ameliorating the amountof weight gained by a companion animal which is fed ad libitumcomprising: administering the combination of 25-OH D, a carotenoid,preferably beta-carotene, Vitamin C and Vitamin E to the animal.Optionally the animal may also be fed the aforementioned combination andat least one further bio-active ingredient selected from the groupconsisting of:

-   -   Vitamin D, Vitamin B2, Vitamin B6, Niacin, Zinc, Copper,        Manganese, Selenium and combinations thereof. Sometimes the        further bio-active ingredients include at least Vitamin D and        Selenium. In some cases, all the further bio-active ingredients        are added.

When using the feed of this invention, the animals may be fed ad libitumand the ill effects normally experienced will be experienced to a lesserdegree or not at all. This eliminates the problems encountered where thehousehold has multiple pets and the owner is trying to manage weight ofsome of the animals.

In preferred embodiments, the combination 25-OH D, carotenoid, vitamin Eand ascorbic acid are added to a basal diet which contains all thenecessary ingredients for complete nutrition, i.e. the combinationprovides a supra-physiological amount of the components. Thus, this canbe distinguished from prior use of one or several of the ingredientswhich are provided in order to merely meet nutritional requirements sothat the animal is not vitamin or nutrient deficient.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows glucose clearance and insulin secretion of broiler breederhens in response to ad libitum feed intake in combination with 25-OHD3+antioxidants/anti-inflammatories. Hens were injected with a singledose of glucose (0.5 g/kg BW) through wing vein after 3 weeks offeeding. Blood samples were collected through cannulation of wing veinat indicated time points after glucose infusion, n=3.

FIG. 2 shows tissue interleukin-1β contents and plasma IL-6 levels ofbroiler breeder hens in response to ad libitum feed intake incombination with 25-OH D3 and antioxidants/anti-inflammatories. Tissuesand blood samples were collected after 10 weeks of the feeding trial.Means with different superscript letters are significantly different(P<0.05), n=3.

FIG. 3. shows tissue STAT-3 activation of broiler breeder hens inresponse to ad libitum feed intake in combination with 25-OH D3 andantioxidants/anti-inflammatories inclusion. Tissues and blood sampleswere collected after 10 weeks of the feeding trial. Means with differentsuperscript letters are significantly different (P<0.05), n=3.

FIGS. 4-6 show pictures taken during the necropsy of broiler hens inresponse to ad libitum feed intake in combination with 25-OH D3 andantioxidants/anti-inflammatories inclusion. Hens were necropsied after10 weeks of the feeding trial.

FIG. 7 shows the effect of dietary supplementation of25-OH-D3+antioxidants/anti-inflammatories on egg production of broilerhens with restricted or ad libitum feed intake.

FIG. 8 shows the effect of dietary supplementation of25-OH-D3+antioxidants/anti-inflammatories on the survival rate ofbroiler hens with restricted or ad libitum feed intake.

FIG. 9 shows the effect of dietary supplementation of25-OH-D3+antioxidants/anti-inflammatories on body weight of broiler henswith restricted or ad libitum feed intake.

FIG. 10 is a series of photos showing the gross morphology of the heartof dead hens with dietary supplementation of25-OH-D3+antioxidants/anti-inflammatories under restricted or ad libitumfeed intake.

FIG. 11 is a series of photographs showing the gross morphology of theheart of dead hens with dietary supplementation of25-OH-D3+antioxidants/anti-inflammatories under restricted or ad libitumfeed intake

FIG. 12 is a series of electrocardiograms (EGCs)demonstrating the effectof dietary supplementation of 25-OH-D3+antioxidants/anti-inflammatorieson EGC of broiler hens with restricted or ad libitum feed intake. Thearrows point to irregularities in the patterns.

FIG. 13 is a series of EGSs showing the effect of dietarysupplementation of 25-OH-D3+antioxidants/anti-inflammatories onarrhythmic ECG pattern of broiler hens with restricted or ad libitumfeed intake.

FIG. 14 shows photos and a graph demonstrating the effect of dietarysupplementation of 25-OH-D3+antioxidants/anti-inflammatories on cardiacfibrosis of broiler hens with restricted or ad libitum feed intake. (atage of 35 weeks)

FIG. 15 are graphs showing the effect of dietary supplementation of25-OH-D3+antioxidants/anti-inflammatories on plasma IL-6 and IL-1βconcentration of broiler hens with restricted or ad libitum feed intake.

FIG. 16 are photos showing the effect of dietary supplementation of25-OH-D3+antioxidants/anti-inflammatories on cardiac cell apoptosis ofbroiler hens with restricted or ad libitum feed intake. (at age of 35weeks)

As used throughout this specification and claims, the followingdefinitions apply: “25-OH D” refers to any form of 25-hydroxyvitamin D(i.e. either 25-OH D2 or 25-OH D3, or mixes thereof). 25-OH D3specifically refers to 25-hydroxyvitamin D3; 25-OH D2 specificallyrefers to 25-hydroxyvitamin D2.

“Vitamin D” means either Vitamin D2, Vitamin D3 or a combination.Vitamin D3 used alone is preferred.

“Companion animal” means dogs, cats, rats, rabbits, ornamental birds,ornamental fish and reptiles.

“Hyperphagia” is excessive eating; the animal does not voluntarily limitits feeding.

“Ameliorating weight gain” means that there is a significant loweramount of weight the poultry would gain when feeding ad libitum withingesting the combination of 25-OH D3 andantioxidants/anti-inflammatories described herein. The animals ingestingthe combination may gain more than those fed a restricted diet.

“Ascorbic Acid” and “Vitamin C” are used interchangeably throughout thespecification and claims.

“Basal diet” means that the food used supplies the animal withsufficient vitamins and minerals so that the companion animal is vitaminand mineral replete.

“25-OH-D3+antioxidants/anti-inflammatories” means the combination of25-OH D3, vitamin E, carotenoid and ascorbic acid, administered in feedas an addition to a basal diet, at a dosage range as set forth in thespecification. Optionally, and preferably, additional bio-activeingredients, selected from the group consisting of: Vitamin D, VitaminB2, Vitamin B6, Niacin, Pantothenic Acid, Folic Acid, Biotin, Zinc,Copper, Manganese, Selenium and combinations thereof are added to the25-OHD3, vitamin E, canthaxanthin and ascorbic acid combination.

“Sudden Death” means that the individual bird died without showingprevious signs or illness or trauma. Upon necropsy, the bird usually hadunrecognized cardiovascular problems.

1. Metabolic Problems

Metabolic problems associated with hyperphagia and which can belessened, reduced or eliminated through use of the feed/premix of thisinvention include:

-   -   a) decreased weight gain and/or no further weight gain;    -   b) clearance of non-esterified fatty acids    -   c) amelioration of plasma dyslipidemia (triglycerides,        sphingomyelin, and ceramide)    -   d) amelioration of triglyceride and ceraminde accumulation in        the liver, leg, breast muscle, and heart    -   e) suppression the tissue pro-inflammatory IL-1β production and        plasma IL-6 concentration    -   f) cardiac protection and enhanced cardiac function through the        up-regulation of the phosphorylation of STAT-3 (signal        transducer and activator of transcription 3) in the heart.    -   g) suppression of the infiltration of immune cells into the        heart    -   h) decreasing the incidence of ascites.

These above-mentioned observed improved conditions result in a loweringof mortality rate, improved insulin signaling, reduced lipotoxicdevelopment and systemic inflammation, and activation ofcardio-protective mechanisms against fuel-overload induced cardiacpathogenesis.

Importantly it has been found in accordance with this invention that thecombination of 25-OH D3, a carotenoid, Vitamin C and Vitamin E canlessen the amount of weight gained when an animal is allowed to eat adlibitum. Thus, this combination can be added to the companion animal'snormal food, and weight management can be easier for the owner.

Along with the lesser weight gain, other benefits associated withdecreasing obesity have also been observed.

2. Cardiovascular Problems

Birds in a flock can experience a sudden death, i.e. the cause of deathis not readily apparent. We investigated this further, as is detailed inEXAMPLE 4. We investigated this phenomenon in flocks fed ad libitum anda restricted diet, with or without the combination of 25-OH D3 andantioxidants/anti-inflammatories. Some of our results are set forthbelow.

It has been found, in accordance with this invention, that thecombination of 25-OH D3, and the antioxidants/anti-inflammatories thatin In birds fed ad libitum, sudden death birds (i.e, birds which haveundergone sudden death) which had been fed with25-OH-D3+antioxidants/anti-inflammatories had higher body weight, butlower relative liver, abdominal fat, and heart weight.

In the birds with sudden death, ad libitum feeding caused cardiacadaptive hypertrophy; and some of the hypertrophic growth may developpathologically into ventricle dilation. As a result, the heart requiresa higher contractility to maintain pumping function to meet the need ofblood supply for oxygen delivery to the peripheral tissues. Thiscondition may have caused heart failure.

Importantly, we found that 25-OH-D3+antioxidants/anti-inflammatoriesdecreased cardiac pathogenic progression and thereby the incidence ofheart failure in birds fed ad libitum. Thus another aspect of thisinvention is the use of 25-OH-D3+antioxidants/inflammatories to reducethe amount of cardiac problems leading to sudden death.

In both restricted and ad libitum fed birds, birds with25-OH-D3+antioxidants/anti-inflammatories exhibited less adaptivehypertrophic growth, supporting the hypothesis that most excessive fuelsmay be partitioned to the muscle, and thereby, hypertrophic growth ofthe heart for increased pumping function cannot meet the need of oxygensupply for higher growth rate (muscle) and thus may provoke cardiacarrhythmia and failure.

25-OH-D3+antioxidants/anti-inflammatories were seen to have thefollowing effects:

-   -   decreased the incidence of cardiac morbidities (dilation,        pericardial effusion, rupture) observed in necropsies of in the        dead birds.    -   Decreased the amount of irregular incidence of ECG patterns    -   Decreased arrhythmia of broiler hens fed ad libitum    -   ameliorated sudden death induced by cardiac morbidities    -   ameliorated cardiac fibrosis in hens fed ad libitum.    -   ameliorated chronic systemic inflammation in hens fed ad        libitum.    -   ameliorated cardiac cell apoptosis in hens with restricted or ad        libitum feed intake.

As these symptoms are expected to involve the same pathways in thecompanion animals as in the observed birds, it can be concluded that thesame pathologies are occurring in the obese companion animals as well.Thus, the 250H D3+antioxidants can protect the cardiovascular system incompanion animals as well.

3. Doses

In one aspect of this invention the combination of 25-OH D3 and theantioxidants/anti-inflammatories are given to animals which are vitaminreplete rather than vitamin deficient. The vitamin replete status ispreferably due to the use of a basal feed which supplies at least theminimum amount of vitamins and minerals for the cat or dog. Thecombination of this invention is thus preferably used in addition to thebasal diet. The lower doses given below are suitable for companionanimals who have reached an ideal weight, as it will assist them fromgaining extra unwanted weight. The medium dosages are suitable foranimals which are overweight or are tending to obesity. The high dosagesare suitable for those animals which have become obese. All thesedosages may be given in combination with a reduced calorie companionanimal food.

25-OH D3:

The amount of 25-OH D3 can range from 15-200 μg/kg food. Preferably, theamount of 25-OH D3 is from 35-150 μg/kg food. For food with a low doseof the combination of the invention, 35 μg per kg 25-OH D3/food ispreferred; for food with a medium dose of the combination, 69 μg per kgfood is preferred; and for food with a high dose of 150 μg feed ispreferred.

Vitamin E:

The amount of Vitamin E can range from 40-400 mg/kg food. Preferably theamount is 80-300 mg/kg food. For food with a low dose of the combinationof the invention, 80 mg/kg Vitamin E is preferred. For food with amedium dose, 150 mg Vitamin E is preferred; for food with a high dose,300 mg/kg Vitamin E is preferred.

Beta Carotene:

The amount of canthaxanthin can range from 1-15 mg/kg feed. Preferablythe amount is 3-12 mg/kg feed. For feed with a low dose of thecombination of the invention, 3 mg/kg canthaxanthin is preferred. Forfeed with a medium dose, 6 mg canthaxanthin is preferred; for feed witha high dose, 12 mg/kg canthaxanthin is preferred.

Ascorbic Acid:

The amount of ascorbic acid can range from 40-400 mg/kg feed. Preferablythe amount is 100-300 mg feed. For feed with a low dose of thecombination of the invention, 100 mg/kg ascorbic acid is preferred. Forfeed with a medium dose, 150 mg ascorbic acid is preferred; for feedwith a high dose, 300 mg/kg ascorbic acid is preferred.

Thus specific preferred feeds of this invention comprise the followingdosages (all amounts are per kg/feed):

Preferred Feed #1:

-   -   25-OH D3: 15-200 μg,    -   Vitamin E: 40-400 mg,    -   Beta Carotene: 1-15 mg; and    -   Ascorbic acid: 40-400 mg.

Preferred Feed #2:

-   -   25-OH D3: 35-150 μg,    -   Vitamin E: 80-300 mg,    -   Beta carotene: 3-12 mg, and    -   Ascorbic acid: 80-300 mg

Preferred Feed #3: (Low dose feed) This feed is preferred for animalswho are at their ideal weight, and is used to help maintain it.

-   -   25-OH D3: 35 μg    -   Vitamin E: 80 mg    -   Beta Carotene: 3 mg    -   Ascorbic acid: 80 mg.

Preferred Feed #4 (medium dose feed) This feed is preferred formoderately to severely obese companion animals:

-   -   25-OH D3: 69 μg    -   Vitamin E: 150 mg    -   Beta carotene: 6 mg    -   Ascorbic acid: 150 mg.

Preferred Feed #5 (high dose feed) This feed is preferred for severelyobese companion animals:

-   -   25-OH D3: 150 μg    -   Vitamin E: 300 mg    -   Beta carotene: 12 mg    -   Ascorbic acid: 300 mg.

The ratios of the above antioxidants/anti-inflammatories relative toeach other may range as follows:

-   -   Vitamin E to Vitamin C may range from 1-10:10-1; preferably        1-5:5-1; and more preferably 1.5:1 to 1:1.5, and most preferably        1:1.    -   Vitamin E or Vitamin C to Beta Carotene may range from 40:1 to        1:1; preferably from 20:1 to 1:1; and more preferably from 10:1        to 1:1

Preferred ratios include the following

Vit E Vit C Beta Carotene 40 40 1 20 20 1 10 10 1

Optional Additional Ingredients:

To each of the feeds listed above, at least one of the additionalingredients may be added. Preferably at least one, and more preferablymore than one of the following ingredients are added. In otherembodiments, all the following ingredients are added:

-   -   Vitamin D3—generally this is present in poultry diets at        approximately 2500 IU per kg feed. In accordance with this        invention, if desired, the amount of Vitamin D is increased to        at least 3000 IU per kg.    -   Vitamin B2: this can be added at 3-25 mg per kg; preferably from        6-20 mg/kg. For low dose feed, 6 mg/kg is preferred. For medium        dose feed, 14 mg/kg is preferred; and for high dose feed 20        mg/kg is preferred.    -   Niacin: this can be added at 25-300 mg per kg feed. Preferably        it ranges from 60-200 mg/kg. For low dose feed, 60 mg/kg is        preferred. For medium dose feed, 120 mg/kg is preferred; and for        high dose feed 200 mg/kg is preferred.    -   Pantothenic acid: this can be added at 10-120 mg per kg feed.        Preferably it ranges from 15-80 mg/kg. For low dose feed, 15        mg/kg is preferred. For medium dose feed, 30 mg/kg is preferred;        and for high dose feed 80 mg/kg is preferred.    -   Folic acid: this can be added at 1-8 mg per kg feed. Preferably        it ranges from 2-6 mg/kg. For low dose feed, 2 mg/kg is        preferred. For medium dose feed, 4 mg/kg is preferred; and for        high dose feed 6 mg/kg is preferred.    -   Biotin: this can be added at 0.05-1.0 mg/kg feed. Preferably it        ranges from 0.2-0.8 mg/kg. For low dose feed, 0.2 mg/kg is        preferred. For medium dose feed, 0.4 mg/kg is preferred; and for        high dose feed 0.8 mg/kg is preferred.    -   Zinc: this can be added at 50-300 mg/kg feed. Preferably it        ranges from 70-250 mg/kg. For low dose feed, 70 mg/kg is        preferred. For medium dose feed, 125 mg/kg is preferred; and for        high dose feed 250 mg/kg is preferred.    -   Copper: this can be added at 5-50 mg/kg feed. Preferably it        ranges from 10-30 mg/kg. For low dose feed, 10 mg/kg is        preferred. For medium dose feed, 20 mg/kg is preferred; and for        high dose feed 30 mg/kg is preferred.    -   Manganese: this can be added at 50-300 mg/feed. Preferably it        ranges from 80-270 mg/kg. For low dose feed, 80 mg/kg is        preferred. For medium dose feed, 150 mg/kg is preferred; and for        high dose feed 270 mg/kg is preferred.    -   Selenium: this can be added at 0.05-0.6 mg/kg feed. Preferably        it ranges from 0.1-0.4 mg/kg. For low dose feed, 0.1 mg/kg is        preferred. For medium dose feed, 0.3 mg/kg is preferred; and for        high dose feed 0.5 mg/kg is preferred.

Premixes can be made to give the above-mentioned doses and preferreddoses. One premix which forms part of this invention is formulated sothat 1 gram of premix is added to one kilogram food, and that theresulting food contains the dosages described in any of the givendosages above. The amounts of the individual ingredients can, of coursebe varied so that one kilogram of premix is added to one metric ton offeed, and that the resulting food contains the dosages described in anyof the given dosages above. There are specific illustrations of this inthe Examples, below.

Further, it is envisioned that the combinations specified herein may beadded to any commercially available dog or cat food, and thus the totalamount of 25-OHD3 and antioxidants/anti-inflammatories present may beequal to the amount originally present in the food plus the addition asspecified herein. It is also envisioned that the 25-OHD3 andantioxidants/anti-inflammatories as specified herein are the soleadditions to a basal diet which contains at least the minimum requirednutrients for cat or dog nutrition.

The following non-limiting Examples are presented to better illustratethe invention

EXAMPLES Example 1

A total of thirty 45-week-old broiler breeder hens (ROSS 308) wereobtained from a commercial flock for the study. A basal broiler breederlaying diet was formulated as shown in Table 1. The calculated nutrientcomposition is shown in Table 2.

TABLE 1 Ingredient composition of the basal broiler breeder layingdiets. Composition %, w/w Corn 66.9 Soybean meal 22.2 Oil fat 1.67 CaCarbonate (ground 6.36 oyster shell) Dicalcium phosphate 1.8 Choline-Cl(70%) 0.1 Mineral Premix¹ 0.1 Copper sulfate 0.05 Vitamin Premix² 0.1¹Mineral premix provided (per kg of diet for treatment groups 1, 2 and3): Cu 18 mg; I 1.1 mg; Fe 80 mg; Mn 150 mg; Zn 125 mg; and Se 0.25 mg.²Refer to Table 2 for further detail.

TABLE 2 Vitamin premix composition (provided per kg of diet) Treatment3: Treatments 1 and 2: 3 = ad libitum feeding + 25- 1 = restrictedfeeding OH-D3 + antioxidants/anti- Vitamin 2 = ad libitum feedinginflammatories A (IU) 10000 12000 D3 (IU) 2500 3000 E (mg) 100 150 K3(mg) 3 5 B1 (mg) 3 5 B2 (mg) 8 14 B6 (mg) 6 8 B12 (mg) 0.03 0.03 Niacin(mg) 60 120 Pantothenic acid 18 30 (mg) Folic acid (mg) 1 4 Biotin (mg)0.2 0.4 C (ascorbic acid) 0 150 (mg) 25-OH-D3 (mcg) 0 69 Canthaxanthin 06 (mg)

TABLE 3 Calculated nutrient composition (%) of the basal broiler breederlaying diets. Composition % w/w Crude protein 16 Crude fat 4.2 Calcium3.1 Sodium 0.16 Total Phosphorus 0.64 Total ME 2910 kcal/kg

Diet was supplemented with or without 25-OH D3 at 69 mcg/kg diet incombination with antioxidants/anti-inflammatories (vitamin E, ascorbicacid, canthaxanthin) and enriched levels of selected vitamins. Hens wererandomly allocated to 3 treatment groups according to feeding regimen(restricted and ad libitum) as follows:

Basal diet—restricted feeding (140 g/day)Basal diet—ad libitum feedingBasal diet—ad libitum feeding+25-OH-D3 at 69 mcg/kgdiet+antioxidants/anti-inflammatories

They were individually-housed in wire cages placed in a controlled roomwith 14 h:10 h light:dark period and at a temperature of 25±3° C. Waterwas available ad libitum. The experimental period was lasted for 10weeks. Three weeks after the feeding trial, some birds were used forrelevant plasma parameter analyses. At the end of experiment, hens wereeuthanized and sacrificed for tissue sample collection for furtherstudies: Necropsy of tissue morphology

-   -   Determination of lipid and sphingolipid profile—serum and        tissues    -   Determination of tissue pro-inflammatory cytokines    -   Determination of insulin resistance    -   Collection of tibia for bone strength analysis    -   Harvesting heart (cardiomyopathy) and skeletal muscle (breast        and thigh) for myopathy analyses.

Example 2 Results and Discussion 25-OH D3 andAntioxidants/Anti-Inflammatories Suppressed Adiposity and Abdominal Fatin Overfed Broiler Hens

Breeder hens are capable of storing large quantities of excess energy(in the form of triglycerides) in the liver, adipose tissue and yolk ofdeveloping oocytes. Lipogenesis (i.e., the conversion of glucose totriglycerides) takes place primarily in the liver of birds and involvesa series of linked, enzyme catalyzed reactions including glycolysis, thecitric acid cycle and fatty acid synthesis. Hepatic lipogenesis issubject to both nutritional and hormonal control and is highlyresponsive to changes in the diet. Adipose tissue serves primarily as astorage site for lipid with little lipogenic activity. Differentiallipogenic capacity of liver vs. adipose tissue in birds is a function ofthe expression of a key transcription factor, sterol regulatory elementbinding protein-1 (SREBP-1). The gene for SREBP-1 is highly expressed inthe liver, but to a much lesser extent in adipose tissue. Moreover, theexpression of a number of lipogenic enzyme genes such as fatty acidsynthase, malic enzyme, acetyl CoA carboxylase, ATP citrate lyase andsteroyl CoA desaturase 1 is directly influenced by SREBP-1.

Breeder hens fed ad libitum accreted more abdominal fat than thoserestricted fed. Dietary supplementation of 25-OHD3 andantioxidants/anti-inflammatories ameliorated the deleterious effect ofad libitum feeding on body and tissue weight, particularly on relativeadipose tissue weight (adiposity) (Table 4). Tibial strength of adlibitum-fed hens was enhanced by 25-OH D3 andantioxidants/anti-inflammatories.

TABLE 4 25-OH D3 and antioxidants/anti-inflammatories on body weight,liver weight, abdominal fat weight and tibial strength of ad libitum-fedbroiler breeder hens Ad libitum feeding + 25-OH-D3 + Ad Antioxidants +Restricted libitum anti-inflam- Pooled feeding feeding matories SEM¹Body weight (kg) 3.67^(c) 4.60^(a) 4.04^(b) 0.26 Liver weight (g)41.4^(b) 48.5^(a) 49.6^(a) 5.6 Liver/body 1.14 1.06 1.23 0.18 weight (%)Abdominal fat 40.9^(c) 185.9^(a) 110.8^(b) 28.9 weight (g) Abdominal fat1.10^(c) 4.01^(a) 2.74^(b) 0.70 weight/body weight (%) Tibial strength32.3^(b) 38.3^(ab) 44.3^(a) 3.57 (kg/cm2) ^(a-c)Within a row, meanswithout a common superscript differ (P < 0.05). ¹Pooled standard errorof the mean.

25-OH D3 and Antioxidants/Anti-Inflammatories Lowered Mortality andImproved Egg Production, Ovarian Morphology and Plasma 17B EstradiolLevel

Secretion of estradiol is the hallmark of successful ovulatoryfollicles. In addition to its role in triggering the preovulatory surgeof gonadotropins, estradiol is an important intra-ovarian growth,differentiation, and survival factor. Inclusion of 25-OH D3 andantioxidants/anti-inflammatories reduced mortality and incidence ofovarian degeneration and ovarian-tumor-like morphology, increased eggproduction and sustained plasma estradiol levels in birds under adlibitum feed intake.

25-OH D3 and Antioxidants/Anti-Inflammatories Ameliorated ImpairedGlucose Clearance and Insulin Sensitivity

Dietary inclusion of 25-OH D3 and antioxidants/anti-inflammatoriesimproves insulin resistance as evidenced by ameliorating fasting plasmaglucose and non-esterifed fatty acid level in overfed hens for 10 weeks(Table 6). In glucose clearance test, lean hens showed a very sharpclearance rate between 30-60 min after glucose infusion, and converselyobese hens had a very sluggish clearance rate between 30-90 min (FIG.1). In insulin secretion, obese hens showed a higher plasma insulinlevel under fasting status and after glucose infusion when compared tolean hens (FIG. 1). Both glucose clearance and glucose-induced insulinsecretion were corrected by 25-OH D3 andantioxidants/anti-inflammatories inclusion in overfed hens for 3 weeks(FIG. 1).

TABLE 5 25-OH D3 and antioxidants/anti-inflammatories on plasma glucose,non-esterified fatty acid (NEFA) and insulin of ad libitum-fed broilerbreeder hens Ad libitum feeding + 25-OH-D3 ++ Ad antioxidants/Restricted libitum anti-inflam- Pooled feeding feeding matories SEM¹After 3 weeks of feeding Plasma glucose 181.5 202.5 188.5 11.9 (mg/dL)Plasma NEFA 0.21^(b) 0.35^(a) 0.25^(b) 0.05 (μmole/mL) After 10 weeks offeeding Plasma glucose 180.6^(b) 212.6^(a) 195.6^(b) 12.7 (mg/dL) PlasmaNEFA 0.35^(b) 0.44^(a) 0.33^(b) 0.05 (μmole/mL) Fasting plasma 1.38 1.151.59 0.21 insulin Glucose-induced 2.66^(a) 1.97b 2.46a 0.36 insulin^(a-b)Within a row, means without a common superscript differ (P <0.05). ¹Pooled standard error of the mean.

25-OH D3 and Antioxidants/Anti-Inflammatories Ameliorated Dyslipidemia

Ad libitum-fed hens elevated plasma triglyceride, ceramide andsphingomyelin levels. However, supplementation of combined 25-OH D3 andantioxidants/anti-inflammatories lowered the level of these lipidmetabolites in the plasma of ad libitum-fed hens (Table 6).

TABLE 6 25-OH D3 and antioxidants/anti-inflammatories on plasmatriacyglycerol, ceramide and sphingomyelin of ad libitum-fed broilerbreeder hens Ad libitum feeding + 25-OH-D3 ++ Ad antioxidants/Restricted libitum anti-inflam- Pooled feeding feeding matories SEM¹After 3 weeks of feeding Plasma 15.6 17.4 14.5 2.9 triacyglycerol(mg/mL) Plasma ceramide 11.5^(b) 18.2^(a) 13.5^(b) 2.83 (nmole/mL)Plasma 0.14^(b) 0.28^(a) 0.16^(b) 0.05 sphingomyelin (μmole/mL) After 10weeks of feeding Plasma 12.75^(b) 15.2^(a) 11.5^(b) 2.2 triacyglycerol(mg/mL) Plasma ceramide 8.1^(b) 12.3^(a) 8.8^(b) 1.65 (nmole/mL) Plasma0.15^(b) 0.22^(a) 0.12^(b) 0.05 sphingomyelin (μmole/mL) ^(a-b)Within arow, means without a common superscript differ (P < 0.05). ¹Pooledstandard error of the mean.

25-OH D3 and Antioxidants/Anti-Inflammatories Reduced Accumulation ofTissue Triglyceride and Ceramide Content

Accumulation of triglyceride and ceramide in the liver, heart and legmuscles was lower in hens fed supplemental 25-OH D3 andantioxidants/anti-inflammatories than in those fed ad libitum (Table 7).

TABLE 7 25-OH D3 and antioxidants/anti-inflammatories on tissuetriacyglycerol and ceramide content of ad libitum-fed broiler breederhens Ad libitum feeding + 25-OH-D3 + Ad Antioxidants + Restrictedlibitum anti-inflam- Pooled feeding feeding matories SEM¹ Triacyglycerol(mg/g tissue) Liver 69.0^(b) 94.8^(a) 79.8^(a) 10.6 Heart 33.1^(b)55.6^(a) 45.6^(a) 6.8 Breast muscle 15.6 18.0 17.4 0.05 Leg muscle30.2^(c) 52.3^(a) 41.2^(b) 6.6 Ceramide (mg/g tissue) Liver 174.5^(b)287.9^(a) 235.0^(a) 52.7 Heart 17.5^(c) 30.2^(a) 23.5^(b) 2.4 Breastmuscle 2.25 2.91 2.52 0.54 Leg muscle 4.12^(b) 7.12^(a) 6.01^(a) 0.85^(a-b)Within a row, means without a common superscript differ (P <0.05). ¹Pooled standard error of the mean.

25-OH D3 and Antioxidants/Anti-Inflammatories Depressed TissueProinflammatory IL-1β Production and Plasma IL-6 Concentrations inOverfed Broiler Hens

Obesity-associated inflammation was ameliorated by dietary 25-OH D3 andantioxidants/anti-inflammatories supplementation as evidenced bysuppressed circulating IL-6 levels and IL-1β production in adiposetissue, liver, leg and breast muscle, and heart (FIG. 2).

25-OH D3 and Antioxidants/Anti-Inflammatories Ameliorated Lipotoxicityin Broiler Breeder Hens Fed Ad Libitum

A central complication of obesity is the development of insulinresistance, which is when insulin is incapable of eliciting postprandialnutrient storage in its primary target tissues, skeletal muscle andliver. Without wishing to be bound by theory, it appears that twoprobable mechanisms may explain how increased adipose stores affectoverall insulin sensitivity throughout the body, contributing to thedown regulation of insulin signaling in peripheral tissues. Firstly, thedelivery of nutrients to cells or tissues is in excess of their storagecapacities and thus this leads to the generation of metabolites thatinhibit insulin action. Of particular importance, lipid derivatives,such as triacylglycerol and ceramide, have been shown to inhibitspecific insulin signaling intermediates, thus blocking postprandialglucose uptake and/or glycogen synthesis. In the case of broiler breederfemales being fed ad libitum, the persistent accumulation of thesemetabolites in peripheral tissues likely contributes to a sustainedstate of insulin resistance throughout the hen and of lipotoxicdevelopment. Secondly, increased adiposity induces a chronicinflammatory state characterized by elevated circulating levels ofproinflammatory cytokines produced from adipocytes or from macrophagesinfiltrating the fat pad. These inflammatory mediators have been shownto antagonize insulin signaling directly, and also to induce catabolicprocesses, thus further increasing the delivery of nutrient metabolitesto insulin-responsive organs.

Overall, excess supply of glucose leading to the formation of excesssaturated fatty acids and therefore accumulation of lipids innon-adipose tissues elevates the cellular levels of—active lipids(sphingolipids) that inhibit the signaling pathways implicated inmetabolic regulation together with activated inflammatory responses andlipotoxic development. In particular, ceramide is a putativeintermediate linking both excess nutrients (i.e., saturated fatty acids)and inflammatory cytokines to the induction of insulin resistance.Moreover, ceramide is toxic in a variety of different cell types and iscapable of damaging the heart, pancreas and vasculature. Moreover,25-hydroxy D3 and antioxidants/anti-inflammatories were effective inameliorating the deleterious effect of metabolic and endocrinedysregulations and pro-inflammatory responses resulting from increasedadiposity occurring in broiler breeder hens fed to satiation.

25-OH D3 and Antioxidants/Anti-Inflammatories Ameliorate CardiacMorbidities, Ascites, and Inflammation in Overfed Broiler Hens

The heart may become dysfunctional due to excess lipid accumulation.That ad libitum feeding promoted triglyceride accumulation in the heartsuggested that increased cardiac fatty acid availability is adaptivelyesterified into triglyceride. In addition, ceramide content of the heartwas also increased as a result of ad libitum feeding. Ceramide is acardiotoxin in lipotoxic cardiomyopathy, which elicited inflammatoryresponses as evidenced by more cardiac infiltration of immune cells.(Table 9).

TABLE 8 25-OH D3 and antioxidants/anti-inflammatories on cardiacresponses of ad libitum-fed broiler breeder hens Ad libitum feeding +25-OH-D3 + Ad antioxidants + Restricted libitum anti-inflam- Pooledfeeding feeding matories SEM¹ Heart weight (g) 14.5^(b) 19.2^(a)17.3^(a) 1.8 Heart/body weight 0.40 0.47 0.43 0.17 (%) Heart septum (HS)2.73 3.01 2.83 0.57 weight (g) HS weight/heart 18.7 14.6 15.7 3.9 weight(%) Right atrium (RA) 1.20^(b) 1.97^(a) 1.72^(ab) 0.38 wall weight (g)RA wall weight/ 8.2 9.6 9.6 1.9 heart weight (%) Right ventricle0.95^(b) 1.51^(a) 1.73^(a) 0.27 (RV) wall weight (g) RV wall weight/6.3^(b) 7.5^(ab) 9.4^(a) 1.5 heart weight (%) Left atrium (LA) 1.17^(b)2.26^(a) 2.02^(a) 0.43 wall weight (g) LA wall weight/ 12.2 11.1 10.93.1 heart weight (%) Left ventricle (LV) 3.78^(b) 4.45^(a) 4.65^(a) 0.34wall weight (g) LV wall weight/ 25.5^(a) 21.7^(b) 25.8^(ab) 2.4 heartweight (%) Incidence of 1/7 5/10 3/10 transudate within pericardium(heart/total) Incidence of heart 1/7 6/10 3/10 ventricle dilation(heart/total) Incidence of 0/7 3/10 1/10 ascites (hen/total) Cardiacimmune 97.9^(a) 127.7^(a) 57.7^(b) 32.4 cell count ^(a-b)Within a row,means without a common superscript differ (P < 0.05). ¹Pooled standarderror of the mean.

Cardiac hypertrophy represents clinically an adaptive response toincreased workload on the heart. However, cardiac responses to neuraland hormonal factors can also incite hypertrophic changes independent ofincreases in afterload or vascular resistance. Fuel overloading-inducedcardiac compensatory growth occurred in broiler breeder hens (Table 8).Cardiac hypertrophy may become maladaptive and eventually develop intopathological conditions, leading to heart failure. These resultssupported the fact that lipotoxic development and hypertrophic growth inthe heart tend to elicit inflammatory responses.

The cardioprotective role of phosphorylated STAT-3 (signal transducerand activator of transcription 3) is becoming increasingly clear inrecent years. Interestingly, combined 25-OH D3 andantioxidants/anti-inflammatories induced greater activation of STAT-3(i.e., phosphorylation of STAT-3) in the heart than restricted-fedbreeder hens (FIG. 3), with the lowest activation being observed in adlibitum-fed broiler breeder hens. The incidence of transudate fluidwithin pericardium, heart ventricle dilation and ascites was alleviatedin ad-libitum-fed breeder hens when supplemented with combined25-hydroxy D3 and antioxidants/anti-inflammatories.

Example 3 Premixes

TABLE 9, below, presents some of the final dose ranges of thecomposition of this invention to be added to the feed.

Ranges Low Medium High Vitamin Unit per kg feed per kg feed per kg feedper kg feed 25-OH-D3 mcg 15-200 35 69 150 Vit E mg 40-400 80 150 300Canthaxanthin mg 1-15 3 6 12 VIt C mg 40-400 100 150 300

TABLE 10. below presents the quantity of premix dosed per 1 kg feed:

0.3 g 0.6 g 1 g 1.8 g 2 g Vitamin Unit premix premix premix premixpremix 25-OH-D3 mg 0.0207 0.0414 0.069 0.1242 0.138 Vit E mg 45 90 150270 300 Canthaxanthin mg 1.8 3.6 6 10.8 12 Vit C mg 45 90 150 270 300

TABLE 11 1 gram premix can be made with the following ingredients:Vitamin Unit 1 g premix 25-OH-D3 mg 0.069 Vit E mg 150 Canthaxanthin mg6 Vit C mg 150 Carrier and others mg to 1000 mg or 1 g

Conclusions

Supplemental 25-OH D3 and antioxidants/anti-inflammatories ameliorateddeleterious effects associated with overfeeding of broiler breederfemales by:

lowering mortality rate and improving ovary function and thereforereproductive performance of overfed broiler breeder hens.improving endocrine (insulin) signalingreducing lipotoxic development and systemic inflammationactivating cardio-protective mechanisms against fuel-overload inducedcardiac pathogenesis

Example 4 Foods

A dry pet food formula

Ingredients % Chicken By-Product Meal 35.40 Corn 30.00 Rice, Brewers10.00 Chicken Fat 6.00 Wheat 5.00 Beet Pulp 4.00 Corn Gluten Meal, 60%3.00 Fish Meal 2.00 Digest (palatants) 1.00 Fish Oil 1.00 DicalciumPhosphate 1.00 Potassium Chloride 0.40 Salt 0.30 Choline Chloride, 60%dry 0.30 Calcium Carbonate 0.20 Vitamin Premix 0.20 Mineral Premix 0.20

To this formula, 25-OHD3, beta carotene, Vitamin C and Vitamin E areadded.

A wet pet food formula

Ingredients % Water 55 Chicken 25 Rice 5 Chicken liver 2 Chicken heart 2Natural flavor 2 Flaxseed 2 Peas (dried) 1.3 Carrots (dried) 1 Apple(dried) 1 Guar Gum 1 Carrageenan 1 Soybean Oil 1 Potassium Chloride 0.5Vitamin premix 0.1 Mineral Premix 0.1

To this formula, 25-OHD3, beta carotene, Vit C and Vitamin E are added.

Example 4 Cardio-Myopathy Trial Example 5 Cardio-Myopathy TrialMaterials and Methods

A total of thirty 45-week-old broiler breeder hens (ROSS 308) wereobtained from a commercial flock for the study. A basal broiler breederlaying diet was formulated as shown in Table 12. The calculated nutrientcomposition is shown in Table 13.

TABLE 12 Ingredient composition of the basal broiler breeder layingdiets. Composition %, w/w Corn 66.9 Soybean meal 22.2 Oil fat 1.67Calcium carbonate 6.36 (ground oyster shell) Dicalcium phosphate 1.8Salt 0.08 Choline-Cl (70%) 0.1 Mineral premix¹ 0.1 Cooper sulfate 0.05Vitamin premix² 0.1 ¹Mineral premix provided (per kg of diet fortreatment groups 1, 2 and 3): Cu, 18 mg; I, 1.1 mg; Fe, 80 mg; Mn, 150mg; Zn, 125 mg; and Se, 0.25 mg. ²Refer to Table 13, below for furtherdetail.

TABLE 13 Vitamin premix composition (provided per kg of diet) Treatments2 and 4 2 = restricted feeding + 25-OH-D3 + Treatments 1 antioxidants/and 3 anti-inflammatories 1 = restricted 4 = ad libitum feeding +feeding 25-OH-D3 + 3 = ad libitum antioxidants/ Vitamin feedinganti-inflammatories A (IU) 10000 12000 D3 (IU) 2500 3000 E (mg) 100 150K3 (mg) 3 5 B1 (mg) 3 5 B2 (mg) 8 14 B6 (mg) 6 8 B12 (mg) 0.03 0.03Niacin (mg) 60 120 Pantothenic acid (mg) 18 30 Folic acid (mg) 1 4Biotin (mg) 0.2 0.4 C (mg) 0 150 25-OH-D3 (mcg) 0 69 Canthaxanthin (mg)0 6

TABLE 14 Calculated nutrient composition (%) of the basal broilerbreeder laying diets. Composition %, w/w Crude protein 16 Crude fat 4.2Calcium 3.1 Potassium 0.44 Sodium 0.16 Total phosphorus 0.64 Total ME2910 kcal/kg

Diet was supplemented with or without Hy⋅D® at 69 mcg 25-OH-D3/kg dietin combination with antioxidants (ascorbic acid, canthaxanthin) andenriched levels of selected vitamins. Hens were randomly allocated totreatment groups according to feeding regimen (restricted and adlibitum) as follows:

-   -   1. Basal diet—restricted feeding (140 g/day)    -   2. Basal diet—restricted feeding+Hy⋅D® (25-OH-D3 at 69 mcg/kg        diet)+antioxidants/anti-inflammatories    -   3. Basal diet—ad libitum feeding    -   4. Basal diet—ad libitum feeding+Hy⋅D® (25-OH-D3 at 69 mcg/kg        diet)+antioxidants/anti-inflammatories

Results:

TABLE 15 Effect of dietary supplementation of25-OH-D3+antioxidants/anti- inflammatories on egg production of broilerhens with restricted or ad libitum feed intake. Restriction + 25- Adlibitum + OH-D3+ 25-OH-D3+ antioxidant/ antioxidant/ anti- anti-Restriction inflammatories Ad libitum inflammatories Dead Dead Dead DeadWhole bird Whole bird Whole bird Whole bird flock flock flock flockflock flock flock flock (n = 68) (n = 19) (n = 70) (n = 11) (n = 80) (n= 58) (n = 79) (n = 47) PROD  51.4 ± 2.5^(a) 47.2 ± 1.9^(a)  51.3 ±2.7^(a) 40.2 ± 1.8^(b) 32.0 ± 2.9^(b)  43.4 ± 1.7^(ab) 34.8 ± 2.7^(b) 45.8 ± 2.1^(ab) YIELD 129.0 ± 5.5^(b) 47.9 ± 2.5^(b) 143.2 ± 5.7^(a)33.3 ± 1.9^(b) 41.1 ± 5.3^(d) 41.5 ± 2.1^(b) 54.7 ± 5.9^(c) 41.1 ±2.0^(b) PROD = Egg production rate (eggs/day/hen, %); YIELD = Egg yield(eggs/hen) Results were expressed with mean ± SEM. Means with differentsuperscript letters are significantly different within the same flock (P< 0.05).

FIG. 7 shows the effect of dietary supplementation of25-OH-D3+antioxidants/anti-inflammatories on egg production of broilerhens with restricted or ad libitum feed intake. From TABLE 15 and FIG.7, it can be concluded that:

-   1. 25-OH-D3+antioxidants/anti-inflammatories improved total egg    yield by promoting survival.-   2. In dead birds, 25-OH-D3+antioxidants/anti-inflammatories had no    effect on egg yield and egg production rate in bird fed ad libitum,    but decreased egg yield and egg production rate in restricted birds.    In the whole flock, however,    25-OH-D3+antioxidants/anti-inflammatories increased egg yield but    not egg production rate.-   3. These results suggested that    25-OH-D3+antioxidants/anti-inflammatories accelerated the    progression into death in restricted birds that are susceptible to    sudden death, and thus acting as a flock culler to exclude the    sudden death-susceptible birds for longer survival and thereby    reduced the flock maintenance cost.

TABLE 16 Effect of dietary supplementation of 25-OH-D3 +antioxidants/anti- inflammatories on mortality of broiler hens withrestricted or ad libitum feed intake and body characteristics of thedead hens Restriction + Ad libitum ++ 25-OH-D3 + 25-OH-D3 + antioxidant/antioxidant/ anti-inflam- Ad anti-inflam- Restriction matories libitummatories (n = 19) (n = 11) (n = 58) (n = 47) Mortality 19/68 11/70 58/8047/79 (dead birds (26.47%) (15.71%) (72.5%) (59.49%) of the total) Bodyweight 3.91 ± 0.090 ^(c)  4.01 ± 0.084^(bc)     4.07 ± 0.083 ^(b) 4.37 ±0.082 ^(a) of the dead birds (kg) Liver weight 89.10 ± 6.79 ^(b)   99.27± 7.92 ^(ab ) 105.52 ± 5.48^(a) 106.70 ± 4.68 ^(a)   of the dead birds(g) Relative 2.28 ± 0.0016 ^(d)  2.47 ± 0.0020 ^(b)   2.59 ± 0.0011^(a) 2.44 ± 0.0009 ^(c) liver weight of the dead birds (g/100 g BW)Abdominal 60.45 ± 4.56 ^(b)   68.54 ± 6.05 ^(b)  148.64 ± 8.45^(c)133.54 ± 9.44^(a)    fat weight of the dead birds (g) Relative 1.55 ±0.0013 ^(d)  1.71 ± 0.0016 ^(c)    3.65 ± 0.0014 ^(a)  3.06 ± 0.0009^(b) abdominal fat weight of the dead birds (g/100 g BW) Heart 18.12 ±0.81 ^(c)    19.93 ± 0.87^(b  )   23.66 ± 0.96^(a) 22.29 ± 0.86^(a)  weight of the dead birds (g) Relative  0.46 ± 0.00023 ^(c)  0.50 ±0.00021 ^(b)    0.58 ± 0.00024^(a)  0.51 ± 0.00018^(b) heart weight ofthe dead birds (g/100 g BW) Results were expressed with mean ± SEM.Means with different superscript letters are significantly different (P< 0.05)

FIG. 8 shows the effect of dietary supplementation of25-OH-D3+antioxidants/anti-inflammatories on the survival rate ofbroiler hens with restricted or ad libitum feed intake

From Table 16 and FIG. 8 it can be concluded that:

-   -   1. 25-OH-D3+antioxidants/anti-inflammatories improved bird        survival rate.    -   2. In birds fed ad libitum, dead birds with        25-OH-D3+antioxidants/anti-inflammatories had higher body        weight, but lower relative liver, abdominal fat, and heart        weight, suggesting that most excessive fuels may be partitioned        to the muscle, and thereby, adaptive hypertrophic growth of the        heart for increased pumping function cannot meet the need of        oxygen supply for higher growth rate (muscle) and thus may        provoke cardiac arrhythmia and failure.

TABLE 17 Effect of dietary supplementation of 25-OH-D3 +antioxidants/anti- inflammatories on carcass characteristics at age of35 and 50 weeks of broiler hens with restricted or ad libitum feedintake Restriction + Ad libitum + 25-OH-D3 + 25-OH-D3 + antioxidant/antioxidant/ anti-inflam- Ad anti-inflam- Restriction matories libitummatories (n = 6) (n = 6) (n = 6) (n = 6) Body wt (kg): at 35 wks 3.57 ±0.19^(b) 3.66 ± 0.15 ^(b) 4.32 ± 0.27 ^(a) 4.36 ± 0.33 ^(a) at 50 wks3.78 ± 0.21^(b) 3.83 ± 0.23 ^(b) 4.62 ± 0.34 ^(a) 4.71 ± 0.38 ^(a) Liverwt(g): at 35 wks 55.35 ± 4.42 ^(b)  57.37 ± 2.27 ^(b)  98.45 ± 4.76^(a )83.37 ± 4.17 ^(b)  at 50 wks 58.67 ± 3.84 ^(b)  59.75 ± 4.67 ^(b)  72.56± 5.14 ^(a)* 68.45 ± 3.42 ^(b)* Relative liver wt (g/100 g BW, %): at 35wks  1.55 ± 0.06 ^(d) 1.57 ± 0.11 ^(b) 2.28 ± 0.10 ^(a) 1.91 ± 0.09 ^(b)at 50 wks 1.55 ± 0.05  1.56 ± 0.14   1.57 ± 0.13*  1.45 ± 0.07* Abdominal fat wt of the dead birds (g): at 35 wks 47.63 ± 2.16 ^(c) 50.57 ± 3.64 ^(c)  123.84 ± 9.84^(a)   95.47 ± 8.12 ^(b)  at 50 wks51.24 ± 2.38 ^(c)  53.12 ± 2.57 ^(c)  147.62 ± 8.55^(c)*  112.47 ± 9.01^(c)  Relative abdominal fat wt (g/100 g BW, %): at 35 wks  1.35 ± 0.11^(c) 1.38 ± 0.06 ^(c) 2.86 ± 0.0 ^(a)  2.19 ± 0.03 ^(b) at 50 wks  1.36± 0.12 ^(c) 1.39 ± 0.50 ^(c)  3.20 ± 0.06 ^(a)*  2.39 ± 0.08 ^(b)* Heartwt (g): at 35 wks 12.69 ± 0.38 ^(b)  12.09 ± 0.31 ^(b)  17.33 ±0.65^(a ) 17.76 ± 0.70 ^(a)  at 50 wks 14.12 ± 0.41 ^(b)   13.88 ±0.0.36 ^(b) 22.21 ± 0.71^(a)*  20.88 ± 0.73^(a)*  Relative heart wt(g/100 g BW, %): at 35 wks 0.357 ± 0.024^(b) 0.331 ± 0.011 ^(b) 0.401 ±0.016^(a)  0.414 ± 0.046^(ab) at 50 wks 0.374 ± 0.025^(b) 0.362 ± 0.024^(b)  0.481 ± 0.015^(a)*  0.443 ± 0.024 ^(ab) Results were expressedwith mean ± SEM. Means with different superscript letters aresignificantly different (P < 0.05) *significant difference vs. age at 35wks.

The low relative liver weight in ad libitum birds at age of 50 weeksappears to be due to ovarian regression developed and thus decreasedestrogen secretion leading to decreased lipid synthesis in the liver foryolk deposition.

FIG. 9 shows the effect of dietary supplementation of25-OH-D3+antioxidants/anti-inflammatories on body weight of broiler henswith restricted or ad libitum feed intake.

Results were expressed as a ratio.

FIGS. 10-11 illustrate the gross morphology of the heart of dead henswith dietary supplementation of25-OH-D3+antioxidants/anti-inflammatories under restricted or ad libitumfeed intake.

Conclusions and annotations from Tables 17 and 18 and FIGS. 9-11:

-   1. Ad libitum feeding caused cardiac adaptive hypertrophy, and some    of the hypertrophic growth may develop pathologically into ventricle    dilation. As a result, the heart requires a higher contractility to    maintain pumping function to meet the need of blood supply for    oxygen delivery to the peripheral tissues and thus may provoke heart    failure.-   2. 25-OH-D3+antioxidants/anti-inflammatories decreased the incidence    of cardiac morbidities (dilation, pericardial effusion, rupture) in    the dead birds.-   3. In both restricted and ad libitum fed birds, birds with    25-OH-D3+antioxidants/anti-inflammatories exhibited less adaptive    hypertrophic growth. This supports the hypothesis that most    excessive fuels may be partitioned to the muscle, and thereby,    hypertrophic growth of the heart for increased pumping function    cannot meet the need of oxygen supply for higher growth rate    (muscle) and thus may provoke cardiac arrhythmia and failure.

TABLE 19 Effect of dietary supplementation of 25-OH-D3 +antioxidants/anti- inflammatories on electrocardiogram (ECG) pattern andarrhythmia of broiler hens with restricted or ad libitum feed intake.See FIGS. 12 and 13 for examples of the EGC patterns. Restriction + Adlibitum + 25-OH-D3 + 25-OH-D3 + antioxidant/ antioxidant/ anti-inflam-Ad anti-inflam- Restriction matories libitum matories (n = 8) (n = 8) (n= 8) (n = 8) ECG pattern 4/8 4/8 1/8 3/8 A at age of 35 wks ECG pattern2/8 3/8 2/8 3/8 B or C at age of 35 wks ECG pattern 2/8 1/8 5/8 2/8 D,E, F, or G at age of 35 weeks ECG pattern 3/8 4/8 0/8 2/8 A at age of 50wks ECG pattern 3/8 2/8 1/8 3/8 B or C at age of 50 wks ECG pattern 2/82/8 7/8 3/8 D, E, F, or G at age of 50 weeks Arrhythmic 0/8 0/8 2/8 1/8ECG pattern at age of 35 wks Arrhythmic 1/8 1/8 4/8 2/8 ECG pattern atage of 50 wks

Conclusions and annotations from Table 19 and FIGS. 12 and 13:

-   -   25-OH-D3+antioxidants/anti-inflammatories decreased irregular        incidence of ECG pattern (pattern D to G) and arrhythmia of        broiler hens fed ad libitum and ameliorated sudden death induced        by cardiac morbidities.

FIG. 14 shows the effect of dietary supplementation of25-OH-D3+antioxidants/anti-inflammatories on cardiac fibrosis of broilerhens with restricted or ad libitum feed intake. (at age of 35 weeks)Means with letters over the bars are significantly different (P<0.05)

Conclusions and annotations from FIG. 14:

-   1. 25-OH-D3+antioxidants/anti-inflammatories ameliorated cardiac    fibrosis in hens fed ad libitum.

FIG. 15 shows the effect of dietary supplementation of25-OH-D3+antioxidants/anti-inflammatories on plasma IL-6 (top graphs)and IL-1β concentration (lower graphs) of broiler hens with restrictedor ad libitum feed intake. Results were expressed with mean±SEM (n=6).Means with different letters over the bars are significantly different(P<0.05)

Conclusions and annotations from FIG. 15:

-   -   25-OH-D3+antioxidants/anti-inflammatories ameliorated chronic        systemic inflammation in hens fed ad libitum.

FIG. 16 shows the effect of dietary supplementation of25-OH-D3+antioxidants/anti-inflammatories on cardiac cell apoptosis ofbroiler hens with restricted or ad libitum feed intake. (at age of 35weeks). Results were expressed with mean±SEM (n=3). Means with differentletters over the bars are significantly different (P<0.05)

Conclusions and annotations from FIG. 16:

-   1. 25-OH-D3+antioxidants/anti-inflammatories ameliorated cardiac    cell apoptosis in hens with restricted or ad libitum feed intake.

Conclusions:

Supplemental 25-OH D3 and antioxidants/anti-inflammatories ameliorateddeleterious effects associated with overfeeding of broiler breederfemales by:

-   -   lowering mortality rate and improving ovary function and        therefore reproductive performance of overfed broiler breeder        hens.    -   improving endocrine (insulin) signaling    -   reducing lipotoxic development and systemic inflammation    -   activating cardio-protective mechanisms against fuel-overload        induced cardiac pathogenesis.

1. A food suitable for a companion animal comprising the combinationcomprising 25-Hydroxy vitamin D (25-OH D), Vitamin C, Vitamin E and acarotenoid selected from the group consisting of: lycopene, astaxanthin,cryptoxanthin, beta-carotene, lutein, zeaxanthin and canthaxanthin.
 2. Afood according to claim 1 comprising: 25-OH D, beta carotene, vitamin Cand Vitamin E and optionally further comprises at least one furtherbio-active ingredient selected from the group consisting of: Vitamin D,Vitamin B2, Vitamin B6, Niacin, Zinc, Copper, Manganese, and Selenium.3. A combination according to claim 1 wherein the amount of Vitamin E orVitamin C to beta carotene may range from 40:1 to 1:1; preferably from20:1 to 1:1; and more preferably from 10:1 to 1:1
 4. A combinationaccording to claim 1 wherein the 25-hydroxy vitamin D is 25-hydroxyvitamin D3 (25-OH D3).
 5. A feed comprising the combination or accordingto claim 1 wherein: the amount of 25-OH D3 is from 15-200 μm/kg theamount of Vitamin E is from 40-400 mg/kg the amount of ascorbic acid isfrom 40-400 mg/kg; and the amount of beta carotene is from 1-15 mg/kg 6.A feed according to claim 1 wherein: the amount of 25-OH D3 is from80-150 μm/kg; the amount of Vitamin E is from 30-300 mg/kg; the amountof ascorbic acid is from 80-300 mg/kg; and the amount of beta-caroteneis from 1-15 mg/kg.
 7. A feed according to claim 1 comprising: 25-OH D3:35-150 m/kg; Vitamin E: 80-300 mg/kg; Beta carotene: 3-12 mg/kg; andAscorbic acid: 100-300 mg/kg.
 8. A feed according to claim 1 comprising:25-0H D3: 35 μg/kg Vitamin E: 80 mg/kg Beta-Carotene: 3 mg/kg; andAscorbic acid: 100 mg/kg.
 9. A feed according to claim 1 comprising:25-0H D3: 69 μg/kg Vitamin E: 150 mg/kg Beta Carotene: 6 mg/kg; andAscorbic acid: 150 mg/kg.
 10. A feed according to claim 1 comprising:25-0H D3: 150 m/kg Vitamin E: 300 mg/kg Beta Carotene: 12 mg/kg, andAscorbic acid: 300 mg/kg.
 11. A method of ameliorating the amount ofweight gained by a companion animal which is fed ad libitum comprising:administering the combination of 25-OH D, a carotenoid, Vitamin C andVitamin E to the animal.
 12. A method according to claim 11 furthercomprising administering at least one further bio-active ingredientselected from the group consisting of: Vitamin D, Vitamin B2, VitaminB6, Niacin, Zinc, Copper, Manganese, Selenium and combinations thereof.